Defects in DNA double‐strand break repair resensitize antibiotic‐resistant Escherichia coli to multiple bactericidal antibiotics

Abstract Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance‐breaker compounds, which inhibit resistance mechanisms and thus resensitize bacteria to existing antibiotics. In the current study, we identify bacterial DNA double‐strand break repair as a promising target for the development of resistance‐breaking co‐therapies. We examined genetic variants of Escherichia coli that combined antibiotic‐resistance determinants with DNA repair defects. We observed that defects in the double‐strand break repair pathway led to significant resensitization toward five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full resensitization. For ciprofloxacin and nitrofurantoin, sensitization manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the promutagenic SOS response. Disruption of double‐strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break‐repair inhibitors can be developed they could resensitize antibiotic‐resistant bacteria to multiple classes of existing antibiotics and may suppress the development of de novo antibiotic‐resistance mutations.


| INTRODUCTION
The emergence of antimicrobial resistance (AMR) poses a significant global health threat, with once trivial bacterial infections becoming increasingly difficult to treat (Bush et al., 2011). AMR has rendered several current antibiotics effectively obsolete, severely limiting infection treatment options (Levy & Marshall, 2004;Ventola, 2015).
There is significant interest in developing combinational drugs that can extend the clinical lifetimes of current therapeutics (Brooks & Brooks, 2014;Tamma et al., 2012). One possibility is the development of "resistance breaking" compounds that increase sensitivity to current antimicrobial therapies (Brown, 2015;Laws et al., 2019).
Many drugs and chemicals are known to induce the SOS response in bacteria, including antidepressants, antivirals, herbicides, and anticancer therapies (Crane et al., 2021;Maier et al., 2018;Mamber et al., 1990). Now there is growing evidence that treatment with certain antibiotics can elevate bacterial mutation rates, potentially increasing the likelihood that antibiotic resistance mutations will appear in bacterial populations (Baharoglu & Mazel, 2011;Gutierrez et al., 2013;Kohanski, Depristo, et al., 2010;Pribis et al., 2019). For any new therapy, it would be desirable to limit the possibility of mutation by (i) narrowing the mutant selection window (Drlica & Zhao, 2007) and (ii) suppressing mutagenesis (Blázquez et al., 2018). The current study identifies bacterial DNA doublestrand break repair (DSBR) as a promising target for the development of such therapies.
Several commonly used bactericidal antibiotics have been shown to damage bacterial DNA either as a direct consequence of their primary mode of action or through secondary effects (Kohanski, Dwyer, et al., 2010). Many forms of DNA damage are lethal to bacteria if left unrepaired (Friedberg et al., 2005). Bacteria have sophisticated systems to repair DNA damage and the action of these repair pathways effectively offsets killing by DNA-damaging antibiotics (Bjedov et al., 2003). Recent studies have demonstrated that the inactivation of bacterial DNA repair pathways can sensitize bacterial cells to multiple antibiotics. Inactivation of recA, a key contributor to DNA repair via homologous recombination, has been shown to reduce minimum inhibitory concentrations (MICs) against the antibiotics ceftazidime (β-lactam; cephalosporin), fosfomycin (phosphonic antibiotic), ciprofloxacin (quinolone), trimethoprim (dihydrofolate synthesis inhibitor), and colistin (polymixin) (Thi et al., 2011). Promisingly, deletion of recA also resensitized a ciprofloxacinresistant strain of Escherichia coli to clinically approachable levels of ciprofloxacin (Recacha et al., 2017). The recA gene is required for the repair of both double-stranded DNA breaks and single-stranded DNA (Del Val et al., 2019). It is unclear whether these antibiotic-sensitizing effects stem from defects in the DSBR or single-strand gap repair (SSGR) pathways. It is also unclear whether these resensitization effects extend to other classes of bactericidal antibiotics. In this study, we aim to address these shortfalls by measuring MICs, examining time-kill kinetics, and determining antibiotic tolerance phenotypes for E. coli strains defective in DSBR and SSGR. Five antibiotics documented as having bactericidal effects were examined: ciprofloxacin (Drlica et al., 2009), nitrofurantoin (McOsker & Fitzpatrick, 1994, kanamycin (Davis, 1987), trimethoprim (Giroux et al., 2017), and ampicillin (Rolinson et al., 1977).
In E. coli, double-strand DNA breaks are primarily repaired through homologous recombination via the RecA protein and RecBCD pathway (Kowalczykowski et al., 1994). Single-strand gaps are predominantly repaired by RecF, RecO, and RecR proteins through their aiding in RecAmediated homologous recombination (Morimatsu & Kowalczykowski, 2003). A third pathway that is utilized under DNA damage conditions is nucleotide pool sanitation (NPS). The NPS pathway removes oxidized nucleotides from the resource pool and thus prevents the insertion of aberrant bases during DNA synthesis (Fowler & Schaaper, 1997). In the absence of one particular NPS enzyme, MutT, insertion of the aberrant base 8-oxo-dGTP into the DNA triggers a form of maladaptive DNA repair that can kill bacterial cells (Giroux et al., 2017). We examined the effects of disrupting MutT alongside the DSBR and SSGR pathways in this study.
DNA damage also induces a mutation-promoting stress-response mechanism called the SOS response (Maslowska et al., 2019). In some circumstances, induction of the SOS response has been observed to increase the frequency of antibiotic-resistance mutations that appear in bacterial populations (Blázquez et al., 2018;Cirz et al., 2005). Among the~40 genes induced during SOS are genes that encode error-prone DNA polymerases known to cause an array of mutations (Goodman & Woodgate, 2013). SOS is induced by RecA* nucleoprotein filaments that form in response to DNA damage (Simmons et al., 2008). Through disruption of recA and other DNArepair genes, there is potential to attenuate SOS mutagenesis. In support of this, previous work has demonstrated that E. coli lacking SOS-induced genes involved in DNA repair (including recA) exhibit significant increases in ciprofloxacin susceptibility (Tran et al., 2016).
Similar findings were again observed in genetic screening and gene expression analysis of SOS response mutant strains in intermediateresistant E. coli (Klitgaard et al., 2018). Enhancing killing and decreasing mutation supply through inhibition of bacterial DNA repair pathways represents a global approach toward the resensitization of antibiotic-resistant bacteria. In this study, we measure the induction of the SOS response by each of the five antibiotics and examine the effects of disrupting DSBR and SSGR on SOS induction.

| Molecular techniques
Plasmid DNA was extracted from E. coli using QIAprep Spin Miniprep kits (Qiagen) as outlined by the manufacturer. E. coli cells were made competent and transformed as previously described (Swords, 2003).
Oligonucleotides used in this study are listed in Table A3 and were synthesized by Integrated DNA Technologies (IDT). Polymerase chain reaction (PCR) amplification was performed using QuickTaq (Roche) as recommended by the manufacturer.
For complementation of the ΔrecA mutants, the plasmid, pHG134 (also referred to as pRecA), was used as described previously . For the complementation of ΔrecB mutants, the plasmid pSRM3 (referred to as pRecB) was constructed by Aldevron (GenBank accession number: OP341514). The recB gene and 200 bp upstream were synthesized and cloned into KpnI/XbaI restriction sites on the pJM1071 plasmid backbone. Complementation plasmids were introduced into the appropriate strains by transformation.
When required, TD tests  were performed following the MIC strip assay. The antibiotic strip was removed from the plate and 5 µl of 40% (w/v) D-glucose solution was then added and left to dry at room temperature. Plates were further incubated overnight at 37°C.
Tolerance was described as the growth of colonies in the zone of inhibition (ZOI) following the addition of glucose.

| Disc diffusion assays and TD tests
Where MIC test strips were unavailable or strain MICs were beyond the strip test range, disc diffusion assays were performed. Cells were grown in 500 µl LB broth for roughly 6 h at 37°C. Then, 100 µl of culture was plated onto an LB agar plate. A sterile 13 mm Whatman ® disc (GE Healthcare) with antibiotic (or compound) was placed in the center of the plate. Final antibiotic/compound concentrations on the discs were as follows unless otherwise stated: 10 mg ampicillin, 1 mg trimethoprim, 3.5 mg kanamycin, 50 µg ML328, 15 µg IMP-1700. Plates were incubated at 37°C overnight. The ZOI surrounding the disc was measured. When required, a modified TD test  was performed following the disc diffusion assay as described above.

| Tolerance regrowth percentage and ZOI area calculations
Regrowth percentages and the area within the ZOI were measured using ImageJ (Schneider et al., 2012). Areas, and subsequent regrowth, under REVITT-MILLS ET AL. | 3 of 31 the MIC strip or antibiotic disc, were excluded from these measurements.
Images of MIC plates were imported, cropped, and aligned using regular ImageJ functions. To select the ZOI, images were subject to thresholding using the "auto-threshold" mean preset function. The ZOI was selected using the "wand" tool and added to the ROI manager. The scale was set to pixels and the area was measured using the "measurements function." For TD image processing, ZOI selection and measurements were conducted as above. To select any colonies within the ZOI, all thresholded regions were selected using the "select all" function and added to the ROI manager. Any bacterial growth already present within the ZOI was included in measurements as follows. The area within the ZOI without bacterial growth was determined by selecting the two ROIs and using the "AND" and measurement functions. TD images were cropped and aligned as above. TD images were subject to thresholding using the "auto-threshold" mean preset function. The ZOI from the MIC plates was added to the TD image and the position was adjusted as needed. Colonies were selected as before using the "select all" function. The area within the ZOI without colonies was measured using the "AND" and measurement functions. Measurements were exported and further processed in excel. Percent regrowth was calculated using the following equation: % regrowth = (TD area without colonies-MIC area without colonies) Zone of inhibition area × 100.
Any area or regrowth under antibiotic strips or discs was not included in the regrowth measurements. The ImageJ macro code used to analyze images is available in Appendix 3.
Resistance to ciprofloxacin commonly develops through the acquisition of mutations in the genes encoding DNA gyrase and topoisomerase IV. Ciprofloxacin has a reduced affinity for these mutant forms of DNA gyrase and topoisomerase IV (Heisig, 1996;Yoshida et al., 1990). In this study, we made use of a ciprofloxacinresistant (Cip R ) derivative of E. coli, CH5741 (Huseby et al., 2017).  (Huseby et al., 2017). It is assumed that double-strand breaks are still formed in this background, but due to reduced target affinity, far higher concentrations of ciprofloxacin would be required.
Reasoning that bacterial DNA repair might offset the killing effects of ciprofloxacin in both sensitive and resistant backgrounds, we examined the sensitivities of E. coli strains that combined defects in the DSBR, SSGR, and NPS repair pathways with ciprofloxacin-resistance mutations.
We determined the MIC for each strain using Liofilchem ® MTS™ (MIC Test Strips). Cells deficient in RecA or RecB were hypersensitive to ciprofloxacin in comparison to the wild-type (ciprofloxacin-sensitive) background ( Figure 1a). This phenotype was rescued upon complementation with recA and recB in trans (Figure 1b), confirming the involvement of both RecA and the DSBR pathway in ciprofloxacin sensitivity. Cells lacking the SSGR protein RecO initially appeared to be more sensitive to ciprofloxacin than wild-type cells, but this was found not to be statistically significant. Deletion of other genes whose products are involved in singlestranded DNA repair (recF and recR) or nucleotide sanitation (mutT) had no significant effect on ciprofloxacin MIC. These findings indicate that DSBR is required for the repair of DNA damage following exposure to ciprofloxacin, in agreement with previous studies (Dörr et al., 2009;Henrikus et al., 2020).
To determine if these findings translated to an antibioticresistant background, the sensitization effect of disrupting DNA repair genes was examined using a ciprofloxacin-resistant (Cip R ) derivative of E. coli, CH5741 (Huseby et al., 2017). Nitrofurantoin has previously gone through a period of decreased use due to fears of toxic side effects, yet it has reemerged as an ISDA-endorsed first line-drug in the treatment of urinary tract infections (UTI; Dason et al., 2011). While this drug is commonly used now, it still shows a low propensity for resistance (Gardiner et al., 2019). The mechanism of action for nitrofurantoin is poorly understood. Studies suggest two mechanisms: (i) inhibition of ribosomes, and consequently, protein synthesis (McOsker & Fitzpatrick, 1994); (ii) direct damage to DNA (Jenkins & Bennett, 1976). Nitrofurantoin is a prodrug. Conversion from the prodrug to active drug form requires nitrofurantoin to be processed intracellularly by the bacterial nitroreductases NfsA and NfsB (Bryant et al., 1981). Resistance to nitrofurantoin is associated with loss-offunction mutations in these two nitroreductases, which results in the drug remaining in the inactive prodrug state (McCalla et al., 1978). In the current study, nitrofurantoin-resistant (Nit R ) strains were constructed through the deletion of nfsA, which encodes the nitroreductase NfsA. At the outset of the study, it was not known whether this mutation would eliminate nitrofurantoin-induced DNA damage or not.
In the nitrofurantoin-sensitive background, both ΔrecA and ΔrecB cells were found to be hypersensitive to nitrofurantoin (Figure 1d).
Deletion of recA resulted in a 15× reduction in MIC from 18.7 ± 2.9 μg/ml (wild-type) to 1.25 ± 0.3 μg/ml. Cells deficient in DSBR (ΔrecB) demonstrated a MIC half that of the wild-type strain (9.7 ± 0.8 μg/ml). This effect was then rescued upon complementation ( Figure 1e). The deletion of the genes recF, recO, or recR, which encode SSGR proteins, also significantly reduced MIC compared to REVITT-MILLS ET AL.
Deletion of recA fully resensitized Nit R cells to the wild-type level (20 ± 2.6 μg/ml). Deletion of recB or recF was even more sensitizing, reducing the MIC to below wild-type levels (14.5 ± 3.7 and 15± 1.2 μg/ml, respectively). Cells lacking other SSGR proteins, RecO and RecR, in addition to MutT showed no significant resensitization.
The potent resensitization effects of recA, recB, and recF mutations strongly suggest that DNA damage still occurs in cells that have developed nitrofurantoin resistance through loss of function mutations in NfsA. Disruption of the DSBR pathway fully resensitizes NfsA-lacking cell activity toward nitrofurantoin.

| Disrupting DSBR enhances killing by kanamycin and trimethoprim
The antibiotics kanamycin and trimethoprim target essential components of the bacterial cell, namely ribosomes and folate biosynthesis (Kohanski, Dwyer, et al., 2010;Visentin et al., 2012). While the primary action of these antibiotics does not directly induce DNA . The means and standard errors of the mean are shown, based on results from at least three biological replicates. Statistical analysis was carried out using twosample Student's t-tests. An asterisk denotes statistical significance (p < 0.05) compared to Cip R . (d) Nitrofurantoin MIC values obtained for isogenic E. coli strains. The means and standard errors of the mean are shown, based on results from at least six biological replicates. Statistical analysis was carried out using Student's t-tests. An asterisk denotes statistical significance (p < 0.05) compared to wild-type (MG1655). (e) Nitrofurantoin MIC values obtained for the E. coli strain wild-type (MG1655) with empty vector (VC; vector control), ΔrecA and ΔrecB mutants with empty vector, and complemented derivatives (pRecA and pRecB, respectively). The means and standard errors of the mean are shown based on results from at least three biological replicates. Statistical analysis was carried out using Student's t-tests. An asterisk denotes statistical significance (p < 0.05) compared to wild-type with empty vector, WT (VC). (f) Nitrofurantoin MIC values obtained for isogenic and Nit R ΔmutT (EKW052). Statistical analysis was carried out using Student's t-tests. The means and standard errors of the mean are shown, based on results from at least three biological replicates. An asterisk denotes statistical significance (p < 0.05) compared to Nit R .
damage, there is now growing evidence that treatment of bacterial cells with bactericidal antibiotics results in the overproduction of reactive oxygen species (ROS) (Dwyer et al., 2014). It has been suggested that treatment with these antibiotics provokes the accumulation of ROS leading to DNA damage (Belenky et al., 2015;Dwyer et al., 2014;Foti et al., 2012;Wang & Zhao, 2009). We, therefore, examined whether cells lacking DSBR, SSGR, and NPS are sensitized to kanamycin and trimethoprim.
We first examined if DNA repair-deficient strains had altered sensitivity to kanamycin by MIC tests. For most strains, MICs were similar to wild-type ( Figure A1a). Disruption of DSBR, SSGR, or NPS did not lead to reduced MICs; MICs were marginally increased in ΔrecO (1.9 ± 0.2 µg/ml) and ΔrecR (1.8 ± 0.2 µg/ml) mutants compared to wild-type (1.2 ± 0.3 µg/ml). We did notice, however, that Trimethoprim is a bactericidal drug that disrupts folic acid biosynthesis by inhibiting the enzyme dihydrofolate reductase (DHFR) (Visentin et al., 2012). Inhibition of DHFR eventually starves the cell of nucleotides (Gleckman et al., 1981). Killing by trimethoprim in many ways mirrors the well-studied phenomenon of thymineless death (Hong et al., 2017). As thymineless death is hypothesized to involve the formation of both double-strand breaks and single-strand gaps (Giroux et al., 2017;Hong et al., 2017), we examined DNA repair-deficient strains of E. coli for sensitivity to trimethoprim. The ΔrecA and ΔrecB mutants showed no significant sensitivity in the MIC assay ( Figure 3a). The SSGR mutant ΔrecO showed sensitivity (0.27 ± 0.04 µg/ml), whereas ΔrecR demonstrated significant resistance (2.36 ± 0.35 µg/ml) to trimethoprim treatment. We also determined cell viability using a more sensitive spot plate dilution assay

| Defects in DSBR reduce tolerance to ampicillin
We next wanted to assess the dependency on DNA repair following treatment with an antibiotic belonging to the β-lactam class. This family of antibiotics targets cell wall synthesis. β-lactams block the transpeptidation of peptidoglycan subunits, reducing cell wall integrity, which increases the frequency of cell lysis events (Kohanski, Dwyer, et al., 2010). Here we chose to focus on the β-lactam, ampicillin. Recent studies have demonstrated that treatment of E. coli with ampicillin increases cellular ROS levels (Dwyer et al., 2014), which are proposed to result in the damage of DNA (Belenky et al., 2015).
Following MIC analysis, we found that the sensitivity of DNA repair-deficient E. coli to ampicillin was not significantly altered in comparison to wild-type ( Figure A2a). Minor differences (less than 1 µg/ml) in MIC were observed for ΔrecA and ΔrecB strains, which were complemented in trans ( Figure A2b). This change in MIC is unlikely to be clinically useful. These findings suggest that DNA repair does not play a significant role in bacterial sensitivity following ampicillin treatment.
Ampicillin and other β-lactam antibiotics are more effective during certain bacterial growth phases, particularly stages of high growth (Tuomanen et al., 1986 (Figures 4a,b and A2d).
Ampicillin tolerance in recA and recB mutants was restored to wildtype levels following complementation in trans (Figure 4c,e). Our findings are in good agreement with previous studies, which have demonstrated that deletion of recA reduced the tolerance of E. coli to ampicillin during early exposure (Kohanski et al., 2007).
We also observed reduced tolerance for ampicillin-resistant 3.4 | DSBR defects suppress induction of the SOS response by ciprofloxacin, nitrofurantoin, and trimethoprim The mutagenic SOS response is triggered by some antibiotics (Blázquez et al., 2018). We qualitatively examined the induction of the SOS response by ciprofloxacin and nitrofurantoin in DNA repair deficient cells using an agar plate-based SOS reporter assay. SOS reporter strains were generated by transformation of DNA repair mutant cells with the plasmid pUA66-P sulA -gfp (Zaslaver et al., 2006), which places gfp under the control of the SOS-inducible promoter P sulA . When exposed to ciprofloxacin, wild-type, ΔrecF, ΔrecO, ΔrecR, and ΔmutT In our hands, no obvious SOS response was detected in any strain following ampicillin treatment up to concentrations of 256 μg/ ml ( Figure A2d). This finding contrasts with previous studies (Blázquez et al., 2012;Thi et al., 2011) which have demonstrated ampicillin-dependent SOS induction in E. coli using similar methods.
We observed that kanamycin treatment did not induce a detectable SOS response in any strain analyzed ( Figure A1d). In agreement with other studies (Baharoglu & Mazel, 2011;Kohanski et al., 2007;Thi et al., 2011), kanamycin treatment does not elicit a detectable SOS response in E. coli. Trimethoprim did induce a clear SOS response signal in both the sensitive and resistant backgrounds (Figure 5e,d).
Disruption of recA eliminated SOS response in both cases. Deletion of recB reduced SOS in both cases. Disruption of SSGR did not reduce SOS, except for a recO deletion in the Tmp R background. We note that the SOS signal was enhanced in the recO deletion in the sensitive background.
3.5 | Putative DSBR inhibitors ML328 and IMP-1700 exhibit off-target effects to potentiate ciprofloxacin activity, sensitizing a multi-drug resistant S. aureus strain to clinically relevant levels of ciprofloxacin (Lim et al., 2019). While these two compounds show early promise, further work is required to confirm the mechanism of action as being inhibition of DSBR.
We examined the biological activity of these two drugs in DNArepair deficient E. coli derivatives using disc diffusion assays (Figure 6a,d). We expected that cells lacking DSBR should not show sensitivity to these compounds, since the drug target (RecB) was no longer present. However, we observed that deletion of recB resulted in an increased sensitivity to both drugs, suggesting that there may be off-target effects. Cells lacking recA were also significantly more sensitive to both compounds. Complementation of recA and recB mutants returned sensitivity to both drugs to wild-type levels, Both the DSBR-inhibiting compounds ML328 and IMP-1700 were constructed using quinolone structural backbones. We reasoned that they each might inhibit DNA gyrase and topoisomerase IV. The authors (Lim et al., 2019) examined this potential activity using purified proteins in bulk biochemical assays and claimed no significant interactions. However, the patterns of strain sensitivity we observed with these two compounds and the strong recB-dependent SOS induction mimicked the results we had obtained for ciprofloxacin. We reasoned that if ML328 and IMP-1700 inhibited DNA gyrase and topoisomerase IV, mutations conferring resistance to quinolones may also confer resistance to these two compounds. We repeated the disc diffusion assays using Cip R DNA repair deficient derivatives ( Figure A6a,b). All strains examined were found to be resistant to both ML328 and IMP-1700 at the concentrations tested. Using broth microdilution assays, the MICs of the Cip R strain for both compounds were determined to be greater than 128 µg/ml ( Figure A6c,d). These findings show that the point mutations gyrA; [S83L, D87N] and parC; [S80I], which typically confer resistance to quinolones, also confer resistance to ML328 and IMP-1700. To confirm which of the threepoint mutations was most important for conferring resistance to these compounds, we repeated disc diffusion assays with isogenic E. These findings suggest that while these two compounds may target DSBR to some degree, in our hands the primary mode of action in E.
coli is inhibition of gyrase and topoisomerase IV.

| Testing of potential inhibitors of RecA and the SOS response
A number of chemical compounds have previously been proposed to inhibit the SOS response via targeting RecA activity and work as resistance-breaking compounds (Alam et al., 2016;Buberg et al., 2020;Lee & Singleton, 2004;Vareille et al., 2007). Of these, we tested two promising compounds. The first was ZnPT which has been  Additionally, activation of the highly mutagenic SOS response was found to be dependent on DSBR, raising the possibility that disrupting DSBR would limit the capacity of bacterial populations to develop further antibiotic resistance mutations. Overall, our findings establish DSBR as a promising target for the design of broad-range resistance-breaking compounds that could be used to dramatically enhance the effectiveness of existing bactericidal antibiotics and suppress the development of antibiotic resistance.

| DSBR as a novel drug target
In this study, we found that disruption of DSBR induced a suite of phenotypes that could lead to more effective antibiotic treatments.
Disruption of DSBR enhanced killing by five disparate classes of antibiotics. Previous studies have shown that recA mutants are highly sensitive to quinolones and other DNA-damaging drugs (Machuca et al., 2021;Maeda et al., 2019;Singh et al., 2010;Thi et al., 2011).
Our observations support and build upon these findings, demonstrating that recB mutants also share this hypersensitivity phenotype.
Importantly, we observed that these sensitization phenomena also extended to drug-resistant E. coli strains, suggesting for the first time that disruption of DSBR via inhibition of RecA or RecBCD might represent a viable strategy for the development of broad-ranging antibiotic resistance breakers.
In this study, we examined E. coli, however, studies by others suggest that disruption of DSBR may improve the killing of other bacterial species. In S. aureus, DSBR pathways promote the survival of both antibiotic-sensitive and -resistant bacteria following exposure to antibiotics such as fluoroquinolones, daptomycin, and nitrofurantoin (Clarke et al., 2021). In a separate study, DSBR-deficient Acinetobacter baumannii were significantly sensitized to colistin, gentamycin, rifampicin, and tigecycline (Ajiboye et al., 2018). For both pathogens, the inactivation of DSBR pathways also increased susceptibility to trimethoprim and sulfamethoxazole (Aranda et al., infections (Ha et al., 2020). This reliance on DSBR for infection is also true for the non-ESKAPE pathogens H. pylori, Salmonella enterica, and Campylobacter jejuni (Amundsen et al., 2008;Cano et al., 2002;Gourley et al., 2017). There are a few exceptions to this finding, for example, DSBR-deficient strains of the acid-fast bacterium Mycobacterium tuberculosis do not have any infectivity defects (Heaton et al., 2014). However, this is likely due to the presence of alternate pathways that can repair double-strand DNA breaks in these cells (Brzostek et al., 2014). Furthermore, in Klebsiella pneumoniae, there are interesting DSB-induced phenotypes (Liu et al., 2020). The anticancer drug bleomycin is known to trigger DSBs. When DSBs were induced in K. pneumoniae either by treatment with bleomycin or by a specific CRISPR-Cas9 catalyzed reaction, this resulted in the formation of a novel "R" biofilm. Yet when another known DSBinducing antibiotic, ciprofloxacin, was tested this novel biofilm was notably absent (Liu et al., 2020). These results show variable cellular response to DSBs in K. pneumoniae. Overall, the bulk of results, in conjunction with our own findings, suggest that DSBR inhibitors may not only potentiate antibiotic activities but also reduce the infection potential of many diverse bacterial pathogens.
At high concentrations, the aminoglycoside kanamycin and the folate inhibitor trimethoprim were more active against recA and recB mutants than against the wild-type strain. These sensitivities were also observed in the respective resistant backgrounds. It remains unclear why the MICs of DSBR-deficient strains were similar to wildtype while also demonstrating notably increased sensitivities to high drug concentrations. One possibility is that high concentrations of these drugs are necessary for initiating DNA damage. Although disruption of DSBR does not significantly resensitize E. coli to these drugs, it does increase the efficacy of killing. It is reasonable to hypothesize that the increased killing observed here may translate to improved infection clearance times in vivo when using combinational DSBR inhibitor-antibiotic therapeutics.
In both the sensitive and resistant backgrounds we observed a strong dependence on both RecA and RecB for tolerance following ampicillin exposure. Tolerance enables bacteria to survive exposure to high levels of antibiotics (Balaban et al., 2019). Importantly, antibiotic tolerance is a key contributor to recalcitrant infections, since these cells survive primary antibiotic treatment (Lewis & Manuse, 2019). Antibiotic tolerance can also act as a precursor for resistance development (Levin-Reisman et al., 2017). Our discovery that ampicillin tolerance in E. coli relies on DSBR suggests that combinational therapy with DSBR-inhibiting drugs may help to eliminate tolerant bacteria.
Mutagenesis is one of the major pathways through which antibiotic resistance develops in bacteria (Blázquez et al., 2018;Maslowska et al., 2019). In DNA-damage settings, including certain antibiotic treatments, the rate of mutagenesis is elevated through activation of the SOS response (Maslowska et al., 2019). It is at concentrations of drug higher than the MIC (where often the SOS response is activated) but within a concentration range where cells are not yet effectively killed, that the development and subsequent selection for drug-resistant mutants frequently occurs. This antibiotic concentration range is known as the mutant selection window (Drlica & Zhao, 2007

| Multiple bactericidal antibiotics induce double-strand breaks
It is well characterized that ciprofloxacin-induced DNA damage predominantly occurs in the form of double-stranded breaks (Drlica et al., 2008(Drlica et al., , 2009Henrikus et al., 2020). For the other antibiotics used in this study, the mechanisms that drive DNA damage remain largely unknown. Throughout this study, we observed several antibiotic-associated phenotypes that were dependent on DSBR.
Kanamycin and trimethoprim treatment at high concentrations was more effective at clearing cells lacking DSBR than wild-type, suggesting at high drug concentrations double-strand DNA breaks occur. We also observed reduced ampicillin tolerance in DSBR deficient strains, suggesting that long-term exposure to ampicillin induces double-stranded breaks. In the case of nitrofurantoin, the survival of cells following antibiotic treatment was strongly dependent on homologous recombination, including the DSBR pathway.
This finding suggests that, in part, the formation of double-stranded breaks is one mechanism by which nitrofurantoin works in E. coli.
Taken together, this study lends further weight to the notion that DNA damage, and in particular double-strand breaks, is a common thread between many bactericidal antibiotics.

| New antibiotic-induced phenotypes associated with DNA gap repair
Single-strand DNA gaps in bacteria are commonly formed postreplication (Friedberg et al., 2005), or via exposure to DNA damaging agents, such as UV (Setlow et al., 1963) and possibly even antibiotics.
If left unrepaired, these gaps can be converted into DSBs, which are highly detrimental to bacterial cells (Cox et al., 2000;Hong et al., 2017 We also examined the importance of NPS in survival following antibiotic treatment. Antibiotic-induced oxidative stress can result in the formation of highly toxic and mutagenic oxidized nucleotide bases (e.g., 8-oxo-dGTP) (Foti et al., 2012), which must be cleared from the nucleotide pool by MutT before they are incorporated into the genome (Fowler & Schaaper, 1997). Although MTH1 (the MutT homolog in humans) holds promise as an anticancer therapeutic target (Samaranayake et al., 2017), disruption of mutT does not appear to be sufficiently important for bacterial cell survival or tolerance to be clinically useful. Furthermore, bacterial cells lacking mutT are highly mutagenic (Fowler & Schaaper, 1997), and following trimethoprim treatment, mutT mutants had an increased relative induction of the mutagenic SOS response. As such, MutT would not be an appropriate target for future drug development.

| Development of DSBR inhibitors: The challenge of off-target effects
Since our work demonstrated multiple benefits of targeting DSBR we investigated the efficacy of the published DSBR inhibitors ML328 (Amundsen et al., 2012) and IMP-1700 (Lim et al., 2019) in E. coli.
Contrary to expectations, cells lacking RecB (the putative target of these compounds) were more sensitive to these two compounds than wild-type. This result implied that there these compounds have offtarget effects in E. coli. Further investigation revealed that these drugs were rendered ineffective in fluoroquinolone-resistant derivatives of E. coli. The putative primary target of these two drugs appears to be DNA gyrase, as a single point mutation (known to increase ciprofloxacin resistance) in gyrA rendered cells resistant to these compounds. In the initial studies on these compounds, bulk biochemical assays determined there was no notable inhibition of E.
However, only a single concentration of inhibitor was used to assess this inhibition activity. It is likely if the drug concentration range were extended further, gyrase inhibition may have been observed.
The ΔrecA mutants were compared to putative inhibitors of RecA and the SOS response (ZnPT; Buberg et al., 2020) and FePcTs (Alam et al., 2016) for sensitivity effects with ciprofloxacin. Previous work has outlined the mechanism by which each of these compounds targets RecA and the subsequent SOS response, resulting in inhibition of the cell hypermutation response (Bunnell et al., 2017).
In our hands, these compounds gave no significant effects when tested only for sensitization compared to a ΔrecA mutation. Effects of 30 μM ZPT + ciprofloxacin suggest greater efficiency of killing at higher ciprofloxacin concentrations, yet no change in MIC from ciprofloxacin-only plates. Use of ZPT at such high concentrations could be unwise as 10 μM ZPT has shown toxicity in human cells (Priestley & Brown, 1980  F I G U R E A3 (a) Representative images of ciprofloxacin minimum inhibitory concentration (MIC) and tolerance (TD Test) plate assays for wild-type (WT), and DNA repair-deficient Escherichia coli strains. (b) Representative images of kanamycin MIC and tolerance (TD Test) plate assays for wild-type (WT), and DNA repair-deficient E. coli strains. (c) Representative images of nitrofurantoin MIC and tolerance (TD Test) plate assays for wild-type (WT) and DNA repair-deficient E. coli strains. (d) Representative images of trimethoprim MIC and tolerance (TD Test) plate assays for wild-type (WT), and DNA repair-deficient E. coli strains.
F I G U R E A5 ML328 (a) and IMP-1700 (b) OD 600 and IC 50 linear regression data for Escherichia coli strains (i and ii) MG1655 (WT; wild-type), (iii and iv) ΔrecA::Kan R (HH020), (v and vi) ΔrecB::Kan R (EAW102). The optical density at 600 nm (OD 600 ) was recorded every 20 min for 18 h. OD 600 measurements were background corrected against noinoculum controls. The means and standard error of the mean are shown from three biological replicates. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of compound with no growth as determined by OD 600 readings. IC 50 values were calculated using data from at least three biological replicates.